•••

HIGHLY EMISSIVE CHIRAL
LANTHANIDE(III) COMPLEXES FOR
LABELLING AND IMAGING

Andrew Timothy Frawley

A thesis submitted for the degree of Doctor of Philosophy

2017

Declaration

The work described herein was undertaken at the Department of Chemistry, Durham University between October 2014 and September 2017. All of the work is my own, except where specifically stated otherwise. No part has previously been submitted for a degree at this or any other university.

Statement of Copyright

The copyright of this thesis rests with the author. No quotation from it should be

published without the author’s prior written consent and information derived from

it should be acknowledged. i

Abstract

Sensitised lanthanide complexes based on macrocyclic chelating ligands have been used extensively to study biological function at a cellular level, due to their very bright and long-lived emission, sharp emission bands and high stability to photodegradation. These photophysical properties, in addition to their excellent chiroptical behaviour, also make these complexes promising candidates for security labelling and as anti-counterfeiting tools.

Novel highly emissive chiral europium(III) complexes based on macrocyclic ligands have been synthesised and their photophysical properties studied. They possess high molar extinction coefficients and are amenable to excitation using commonly available light sources. These complexes have been resolved by chiral HPLC and the circularly polarised luminescence (CPL) spectra of their enantiomers recorded. They exhibit strong circularly polarised emission in response to excitation using near ultra-violet light, and are stable to thermally-activated racemisation.

A simple off-the-shelf camera set up has been developed which is capable of

discriminating ‘real’ europium(III) emission from emission from a ‘fake’ marking,

based on emission lifetime and wavelength. Additionally, chiroptical discrimination has been achieved using a custom built microscope incorporating a quarter-wave plate and linear polariser.

The solvent dependent emission behaviour of a series of C₃-symmetric lanthanide(III) complexes has been studied, demonstrating that the form of the total emission and CPL is extremely sensitive to minor changes in the outer solvation sphere of the complex.

Finally, macropinocytosis has been identified as the mechanism of cell uptake of this family of complexes in NIH-3T3 cells, and the internalisation and subsequent sub-cellular localisation has been shown to be dependent on complex chirality.

ii

Acknowledgements

I would like to thank the following people for making this work possible: My supervisor, Prof. David Parker, for giving me the opportunity to carry out this project and for his ideas, guidance and support over the last 4 years.

Special thanks must go to Dr. Robert Pal for his help with all things both living and optical, specifically cameras, microscopes and cells.

Thank you to the following members of the Chemistry Department for their assistance: Dr. Alan Kenwright, Dr. Juan Aguilar, Catherine Heffernan and Dr. Raquel Belda-Vidal for NMR spectroscopy; Dr. Jackie Mosely, Pete Stokes and Dr. Dave Parker for mass spectrometry; Dr. Aileen Congreve and Lenny Lauchlan for HPLC training and for letting me loose in the HPLC lab; Dr. Lars-Olof Pålsson for assistance with low temperature photophysical measurements; Annette Passmoor, Gary Southern and all the stores and technical team for keeping us supplied with equipment and chemicals; the lab attendants for the daily supply of tea and coffee, and specifically to Claire for gently encouraging me to tidy my desk every so often. Thanks also to Dr. Chris Ottley (Dept. of Earth Sciences) for ICP-MS analyses.

Thanks to all the Parker Group members, past and present. In particular I’d like to thank Steve, Brian, Kanthi, Cidalia, Nicola, Matthieu, Kevin, Alex, Martina, Emily, Katie, Sergey, Edward, Alice, Laura, Ryan and Hannah for sharing their knowledge and keeping me going over the years.

I would also like to acknowledge Durham University for the award of a Durham Doctoral Scholarship which has allowed me to pursue my postgraduate studies.

Thanks to all the leaders, parents and young people at 19^th Durham Scout Group for providing plenty of fun and adventure (and distraction from chemistry). A special

thank you to Siân, Will, Evelyn and Nicholas Greeves for being my ‘Durham family’ for the last 7 years. I really can’t thank you enough for everything you’ve done.

Finally, the biggest thanks to Mum, Dad and Helen for their unwavering support in everything I do.

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List of Abbreviations

α₁-AGP 12-N₄ 9-N₃ A431 AAT alpha-1-acid glycoprotein 1,4,7,10-tetraazacyclododecane 1,4,7-triazacyclononane human epidermoid carcinoma cell line alpha-1-antitrypsin

• AC
• alternating current

acac ADP AFM AMP ATP acetylacetonate adenosine diphosphate atomic force microscopy adenosine monophosphate adenosine triphosphate 2,2’-bipyridine-6,6’-dicarboxylate back energy transfer bda BET BINAP BINOL BODIPY BP
2,2'-bis(diphenylphosphino)-1,1'-binaphthyl 1,1'-bi-2-naphthol boron-dipyrromethene band pass

• br
• broad (NMR)

• BSA
• bovine serum albumin

charge-coupled device Cambridge Crystallographic Data Centre circular dichroism
CCD CCDC CD CHO CIE
Chinese hamster ovary Commission internationale de l'éclairage (International Commission on Illumination)

CPEL CPL circularly polarised electroluminescence circularly polarised luminescence

• chiral stationary phase
• CSP

iv

• CW
• continuous wave

cyclen d, dd, ddd DC
1,4,7,10-tetraazacyclododecane doublet, doublet of doublets, etc. (NMR) direct current
DCM dfppy DFT dichloromethane difluorophenylpyridine density functional theory
DMEM DMF DMSO DOTA dpa
Dulbecco's modified Eagle's medium N,N-dimethylformamide dimethylsulfoxide 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid dipicolinic acid, pyridine-2,6-dicarboxylic acid 1,1'-bis(diphenylphosphino)ferrocene 1,3-dipyridylbenzene dppf dpyb DSLR DTPA ED digital single lens reflex diethylenetriaminepentaacetic acid electric dipole

• ee
• enantiomeric excess

퐸_푇^푁

Reichardt's normalised solvent polarity

• equivalents
• eq.

• ESI
• electrospray ionisation

• eT
• electron transfer

• ET
• energy transfer

facam FOV
3-trifluoroacetylcamphorate field of view
FRET FWHM GCMS HEPES hfac
Förster/fluorescence resonance energy transfer full width half maximum gas chromatography mass spectrometry 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid 1,1,1,5,5,5-hexafluoroacetylacetonate

v

• hfbc
• 3-heptafluorobutyrylcamphorate

• HP-DO3A
• 10-(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane-1,4,7-

triacetic acid

HPLC HRMS IC high pressure/performance liquid chromatography high resolution mass spectrometry internal conversion

IC₅₀

half-maximal inhibitory concentration inductively coupled plasma mass spectrometry internal charge transfer
ICP-MS ICT

• IR
• infra-red

• ISC
• intersystem crossing

iTTL LCMS LED through the lens metering (camera flash unit) liquid chromatography mass spectrometry light emitting diode

• Ln
• lanthanide

• LP
• long pass

LSCM LTG laser scanning confocal microscopy LysoTracker Green

• m
• multiplet (NMR)

m.p.

mCPBA

MD melting point meta-chloroperbenzoic acid magnetic dipole
MOPS MRI Ms
3-(N-morpholino)propanesulfonic acid magnetic resonance imaging methanesulfonyl
MTG NIH-3T3 NMR NOTA PBS
MitoTracker Green mouse skin fibroblast cell line nuclear magnetic resonance 1,4,7-triazacyclononane-1,4,7-triacetic acid phosphate buffered saline

vi pda PEM PET phen PhMoNa PMMA PMT ppm ppy PVC Q
1,10-phenanthroline-2,9-dicarboxylic acid photoelastic modulator positron emission tomography phenanthroline phase modulated nanoscopy poly(methyl methacrylate) photomultiplier tube parts per million phenylpyridine poly(vinyl chloride) quencher

• QR
• quick response

QToF Squadrupole time of flight (mass spectrometry) singlet (energy level)

• s
• singlet (NMR)

SEM SOM Tscanning electron microscopy single organic molecule triplet (energy level)

• t
• triplet (NMR)

TBAF td tetrabutylammonium fluoride triplet of doublets (NMR)

• 2,2,2-trifluoroethanol
• TFE

THF TLC TQD TTA UPLC UV tetrahydrofuran thin layer chromatography triple quadrupole (mass spectrometry) 2-thenoyltrifluoroacetonate ultra performance liquid chromatography ultra violet

• YAG
• yttrium aluminium garnet, Y₃Al₅O₁₂

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Table of Contents

Abstract........................................................................................................................ii Acknowledgements..................................................................................................... iii List of Abbreviations ................................................................................................... iv CHAPTER ONE : INTRODUCTION..................................................................................1
1.1 Introduction to lanthanide luminescence.....................................................1
1.1.1 Overview ................................................................................................1 1.1.2 Emission lifetime....................................................................................5 1.1.3 Excitation of lanthanides........................................................................6 1.1.4 Quenching processes .............................................................................7 1.1.5 Photoluminescence quantum yield and brightness ..............................8 1.1.6 Ligands for Ln(III) complexes..................................................................9
1.2 Introduction to circularly polarised luminescence......................................18
1.2.1 Theoretical background .......................................................................18 1.2.2 Instrumentation for the detection of CPL............................................21
1.3 CPL-active organic and transition metal systems........................................23
1.3.1 Single Organic Molecule (SOM) systems .............................................23 1.3.2 Supramolecular aggregates .................................................................27 1.3.3 Transition metal complexes.................................................................29
1.4 Chiral lanthanide complexes .......................................................................32
1.4.1 Design and control of chirality .............................................................32 1.4.2 Ln(III) complexes as CPL probes for biological analytes ......................40
1.5 Cell imaging with lanthanide complexes.....................................................51

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1.5.1 Design considerations ..........................................................................51 1.5.2 Examples of sub-cellular localisation profiles......................................52 1.5.3 Cell uptake mechanisms ......................................................................57
1.6 Lanthanides in security labelling .................................................................61 1.7 Project Aims and Outline.............................................................................69
CHAPTER TWO : CHIRAL Eu(III) COMPLEXES FOR SECURITY LABELLING ...................71
2.1 Requirements for a security label ...............................................................71 2.2 Synthesis of target systems.........................................................................72
2.2.1 Synthesis of [EuL¹]................................................................................73 2.2.2 Synthesis of [EuL²]................................................................................81
2.3 Photophysical properties of [EuL¹] and [EuL²] ............................................84 2.4 Complex chirality and resolution by chiral HPLC.........................................88 2.5 CPL characterisation....................................................................................94 2.6 An alternative strategy for the synthesis of enantiopure carboxylate complexes ............................................................................................................106

2.7 Racemisation kinetics................................................................................114 2.8 Development of time-gated photography in security labelling................121 2.9 Time-gated chiral contrast microscopy.....................................................133 2.10 Conclusions and Future Work ...................................................................137
CHAPTER THREE : SOLVENT EFFECTS IN Ln(III) EMISSION .......................................140
3.1 Introduction...............................................................................................140 3.2 Solvent effects in the emission and CPL of [EuL²].....................................142
3.2.1 Solvent effects in the total emission of [EuL²]...................................142 3.2.2 Solvent effects in the CPL of [EuL²]....................................................148 3.2.3 Chiral additives...................................................................................152

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3.3 Solvent effects in the total emission and CPL of [EuL⁶] ............................156
3.3.1 Solvent effects in the total emission of [EuL⁶]...................................157 3.3.2 Solvent effects in the CPL of [EuL⁶]....................................................159 3.3.3 Specific solvent effect of chlorinated solvents ..................................162
3.4 A combined emission and NMR study into solvent effects in the [LnL⁷] series ...................................................................................................................167

3.5 Conclusions and Future Work ...................................................................182
CHAPTER FOUR : CELL UPTAKE OF CHIRAL Eu(III) COMPLEXES ...............................184
4.1 Introduction...............................................................................................184 4.2 Cell uptake mechanism of [EuL⁴]...............................................................186 4.3 Effect of chirality on cell uptake and sub-cellular localisation..................191
4.3.1 Cell uptake of enantiopure complexes ..............................................193 4.3.2 Sub-cellular localisation of enantiopure complexes..........................198
4.4 Conclusions and Future Work ...................................................................205
CHAPTER FIVE : EXPERIMENTAL METHODS.............................................................208
5.1 General Procedures...................................................................................208 5.2 HPLC analysis.............................................................................................209 5.3 Optical measurements ..............................................................................211 5.4 Time-gated photography...........................................................................212 5.5 Circularly polarised microscopy ................................................................212 5.6 Preparation of PMMA films.......................................................................213 5.7 Cell culture protocols ................................................................................213 5.8 Confocal microscopy and cell imaging ......................................................215 5.9 Synthetic procedures.................................................................................217
REFERENCES .............................................................................................................256